DNA double-strand breaks (DSBs) arise from normal cellular processes and from exogenous sources, such as ionizing radiation. Cells have multiple mechanisms to repair DSBs, including nonhomologous end-joining (NHEJ) and homologous recombination (HR). Lack of repair can lead to genetic loss and cell death, whereas misrepair of DSBs can lead to genomic rearrangements such as chromosomal translocations that are associated with tumorigenesis. Sequencing of breakpoint junctions from patients indicates that translocations primarily arise by some type of NHEJ. A relatively well-characterized canonical NHEJ pathway has been defined, but NHEJ can also occur without canonical NHEJ components, and is loosely termed alternative NHEJ (alt-NHEJ). The specific aims are: 1. To further elucidate mechanisms and the role of alt-NHEJ in mouse cells. Alt-NHEJ is reported to be the primary mechanism for chromosomal translocation formation in mouse cells. We will determine what factors promote and suppress translocations and alt-NHEJ, with a focus on factors that are implicated in controlling DNA end resection. The development of an end resection assay is also proposed. Further, the role of the major alt-NHEJ DNA ligase, Lig3, will be examined in vivo. 2. To develop a clinically relevant translocation system in human stem cells. Specifically, we will induce the Ewing sarcoma EWS-FLI1 translocation in genetically unmodified mesenchymal precursor and stem cells and determine its acute consequence on cell proliferation. Intra-locus deletions within the FLI1 locus will also be interrogated. Both typs of genome destabilizing repair involving 2 DSBs (translocations, deletions) will be investigated relative to genome stabilizing repair of a single DSB. 3. To elucidate mechanisms of chromosomal translocation formation and intra-locus deletions in human cell mutants. Human cells have been reported to have higher canonical NHEJ activity than rodent cells. A key question will be how canonical and alt-NHEJ participate in translocation formation, determined using human cell mutants in both pathways, with a focus on DNA ligase mutants. Intra-locus deletions and single-break DSB repair will also be examined, providing a comprehensive analysis of chromosomal DSB repair in human cells and the genetic requirements. The effect of Lig3 loss on the radiation response will also be assessed.